Clinical photodynamic therapy (PDT) dosimetry is limited. Typically a light dose is measured, but no account is explicitly taken for tissue optical properties or drug concentration. These and other tissue properties can be derived from optical absorption and emission measurements. In this project we will develop, validate, and exploit two instruments to be used in-situ to determine tissue optical properties such as absorption, scattering, fluorescence, and temporal fluctuations averaged over macroscopic tissue volumes in animal models and in human clinical trials. One instrument is a contact probe with multiple source-detector separations used to rapidly determine underlying tissue obsorption and fluorescence across a broad spectral range. The other instrument is a non-contact probe with multiple source-detector separations used to determine tissue absorption and scattering at discrete wavelenghts in the near-infrared; it also provides a useful measure of tissue blood perfusion. Our long-range project goal is to show that such optical techniques, sensitive to macroscopic tissue volumes, can improve PDT dosimetry. The initial validation of these new experimental tools will utilize tissue phantoms. Experimentation will then continue in animal models in which, for example, we will study the changes in tissue optical properties accompanying PDT-induced necrosis. Finally, a range of pilot studies in human tissues will be initiated. Knowledge about tissue optical properties makes possible the assignment of true light dose as a function of depth. Knowledge about tissue fluorescence before and after the PDT treatment provides information about drug concentration and drug utilization. The optical property information will be used to define the average blood oxygen saturation, hemoglobin concentration, and blood flow properties of the tissue samples. These measured variables will be correlated with tissue necrosis and tissue depth of necrosis, so that models for PDT threshold dosimetry can be tested. The research of Project 3 will investigate microscopic properties of these same tissue types to determine the degree to which threshold doses and physiological properties obtained by bulk measurement, remain valid in tissues exhibiting microscopic heterogeneities in concentration and oxygenation. Finally, in collaboration with Projects 1 and 2 we will assess dose heterogeneity hypoxia, and flow in ongoing human intraperitoneal (IP) clinical trials. This quantitative information may improve the optimization of PDT treatments. Rapid, minimally invasive, in-situ optical diagnostics for PDT has enormous clinical potential if they can be shown to work.